Ecosystems and Human Health - Chapter 5 doc

Dioxin TCDD toxicity Dioxins are a family of compounds of which TCDD 2,3,7,8-tetrachlorod-ibenzo-p-dioxin has received the most public attention.. Exposure was well documented and serum

chapter five

Halogenated hydrocarbons and halogenated aromatic hydrocarbons

Introduction

Halogens are the related elements chlorine (Cl), bromine (Br), fluorine (F), and iodine (I) They may exist as gases (Cl2,F2), a liquid (Br2), or a solid (I2) Halogenated hydrocarbons, also known as organohalogens, are a group of organic compounds of diverse structure to which one of these halogens has been attached The core structure may be either simple, consisting of one or two carbons, or more complex and aromatic (halogenated aromatic hydro-carbons or HAHs) Because they have been implicated almost universally in toxic reactions in mammals and lower species (including carcinogenesis in some), it is appropriate to consider them as a group despite their chemical diversity Polycyclic aromatic hydrocarbons (PAHs) are multiringed, planar chemicals that share many of the same toxicological properties as HAHs

Early examples of toxicity from halogenated hydrocarbons

One of the oldest and simplest of these compounds is carbon tetrachloride (CCl4), which was used extensively as a solvent and a dry-cleaning agent until its hepatotoxic nature was discovered In fact, it was used originally

as a treatment for hookworm in humans and domestic animals and as a component in fire extinguishers These uses may still exist in some parts of the world CCl4 owes its toxicity to the fact that it is converted in the liver

to carbon trichloride (CCl3), which is a free radical capable of inducing peroxidation of lipid double bonds and poisoning protein-synthesizing enzymes Older, halogenated hydrocarbons include trichloroethylene and the anesthetics halothane and chloroform (now abandoned because of its toxicity) Figure 21 shows the variety of structural formulae included in this class of compounds The earliest form of poisoning associated with a halogen was probably “bromism,” a condition resulting from the use or abuse of

sodium or potassium bromide as a sedative and sleeping potion in the early 1900s Symptoms included severe headache, stupor, delirium, cardiac prob-lems, very bad breath (from the bromine), and an acneform skin rash of the type now called “chloracne.” The word bromide is used now to indicate a soothing but meaningless statement of the sort frequently uttered by certain politicians

Physicochemical characteristics and classes

of halogenated hydrocarbons

The characteristics of halogenated hydrocarbons that make them useful for

a variety of applications are generally the same ones that make them haz-ardous to the environment and to humans These include:

1 High lipid solubility,

2 Ability to survive heat >800°C,

3 High resistance to chemical breakdown, and

4 Toxicity to microorganisms These agents are used for a variety of purposes (see below)

Antibacterial disinfectants

Hexachlorophene has been used for many years as a surgical scrub and, in

a 3% solution, as a hospital disinfectant It is also used as the active ingredient

in deodorant soaps In the late 1960s, a change was proposed in U.S.FDA regulations to permit the use of hexachlorophene as an antifungal wash for fruit and vegetables

Figure 21 Structural formulae of a number of halogenated hydrocarbons.

O

O Cl

Cl

Cl

Cl

PBBs

X = Br or H

(210 possible structures for each)

TCDD or 2,3,7,8-tetrachlorodibenzodioxin is one of 75 possible dioxins

In light of the then-recent thalidomide tragedy, extensive testing was required for approval to be granted Rats fed high levels of hexachlorophene developed weakness, ataxia, paralysis, and evidence of a type of brain pathology known as status spongiosus, indicative of axonal degeneration

In 1971, a study was done in which infant monkeys were washed daily for

90 days in 3% hexachlorophene Neurological symptoms were observed and status spongiosus was seen in all specimens at post-mortem Significant blood levels have been detected in infants washed with 3% hexachlorophene, and those with severe diaper rash, burns, or congenital skin disorders are especially prone to absorb it, as the natural permeability barrier has been disrupted (see Chapter 1) Autopsies of infants dying from a variety of causes and who had received high exposures showed evidence of status spongiosus

In 1972, hexachlorophene was accidentally added in high concentration to baby powder during its manufacture in France Forty-one deaths of infants and young children were attributed to this error A few years later, Dr Hildegard Halling, a Swedish physician, published a report indicating that nurses who washed frequently in hexachlorophene (10 to 60 times daily) had a higher incidence of birth defects in their offspring (25/460 births) than those who did not (nil/233 births) Although this clinical study was criticized for design flaws, other studies with rats have revealed teratogenic effects This product is no longer used in nurseries in North America and pregnant women are advised to avoid it

Herbicides

This group includes 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophe-noxyacetic acid (2,4,5-T), and dioxins such as 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD = -dioxin) Agent Orange, used as a defoliant in Vietnam, was equal parts of 2,4,-D and 2,4,5,-T and contained TCDD as a contaminant One

of the best-documented human exposures to dioxin was the explosion at Seveso, Italy (see Chapter 2) Hundreds of individuals suffered from chloracne, which is the hallmark of toxicity of halogenated hydrocarbons

The herbicides 2,4,-D and 2,4,5-T are used to control broad-leafed plants along highways, railways, and utility rights-of-way They are hormonal growth promoters and force plants to consume energy at a greater rate than

it can be replaced The humans at greatest risk of toxic exposure are the workers who apply the sprays, and poisoning from dermal and respiratory absorption has occurred as well as from accidental ingestion Signs and symptoms include peripheral neuritis, muscular weakness, and chloracne Although 2,4,5-T is a weak teratogen in some animals, it is the presence of TCDD (or dioxin) that is the greatest source of public concern

Dioxin (TCDD) toxicity

Dioxins are a family of compounds of which TCDD (2,3,7,8-tetrachlorod-ibenzo-p-dioxin) has received the most public attention There is significant

species variation in TCDD toxicity, with the guinea pig being most sensitive (LD50 1 µg/kg) and the rat quite insensitive (LD50 22 µg/kg) There are other toxic manifestations of dioxin toxicity

Hepatotoxicity

All species show enlarged livers, and microsomal monooxygenase enzyme induction occurs in most Rats develop fatty livers with triglyceride deposi-tion Hepatic fibrosis has been reported in humans People exposed at Seveso had elevated serum enzyme levels (serum glutamic-oxaloacetic transami-nase [SGOT] and serum glutamic-pyruvic transamitransami-nase [SGPT]), indicating liver damage, for several weeks after the accident

Porphyria

Porphyrins are pigments widely distributed in nature and are present in the body as by-products of heme synthesis which is required for the for-mation of hemoglobin, myoglobin, and cytochromes Heme is ferrous pro-toporphyrin IX Hematin, the iron-containing molecule in catalase and per-oxidases, is ferric protoporphyrin IX The rate-limiting step in the synthesis

of heme is ALA synthetase (ALA = gamma-aminolevulinic acid) Dioxin significantly increases the levels of ALA synthetase and, hence, ALA levels and the synthesis of porphyrins This is not enzyme induction, but probably

is due to interference with a feedback control system The excess porphyrins are excreted in the urine, giving it a port wine color, and they are deposited

in the skin, producing pigmentation Because porphyrins are photoreactive,

a condition known as porphyria cutanea tarda develops, characterized by photosensitivity, blistering, fragility of the skin, pigmentation, and hirsut-ism (hairiness)

Congenital defects in porphyrin metabolism cause the same syndrome and this has been suggested as the explanation for the werewolf and vampire myths of Europe (characterized by hirsutism and the avoidance of sunlight) There is even an explanation of why drinking blood might have a therapeutic effect Heme is the feedback substance that turns off ALA systhetase Absorp-tion of sufficient heme might thus inhibit porphyrin synthesis In the late 1950s, an extensive outbreak of porphyria cutanea tarda occurred in Turkey during a famine as the result of consuming seed grain treated with hexachlo-robenzene as an antifungal agent A simplified scheme of the steps involved

in porphyrin synthesis is shown in Figure 22

Chloracne

This skin disorder is typified by rash, cysts, and hyperpigmentation and is the hallmark of poisoning with all halogenated hydrocarbons It was the predominant toxic manifestation at Seveso

Cardiovascular effects

A 10-year mortality study of the population exposed to TCDD after the Seveso explosion of 1976 revealed significantlly increased mortality from cardiovascular events

Carcinogenicity

Dioxin is a potent hepatocarcinogen in mice and to a lesser extent in rats There is a latency period before the emergence of liver tumors Evidence of cancer in several studies of Vietnam veterans, for whom claims of increased incidence of cancer have been made, has been inconclusive

A retrospective cohort study was conducted by scientists at the (U.S.) National Institute for Occupational Safety and Health (NIOSH) on 5172 workers at 12 U.S plants in which TCDD was a chemical contaminant of the manufacturing process Exposure was well documented and serum TCDD levels were obtained from 253 workers The mortalities from several cancers previously associated with TCDD (stomach, liver, and nasal cancers, Hodgkin’s disease and non-Hodgkin’s lymphoma) were not significantly different from the overall population, but the incidence of all cancers taken together was slightly but significantly increased In a subcohort of 1520 workers with more than 1 year of exposure and more than 20 years of latency, mortality from soft tissue sarcoma was significantly higher than for the general population The authors concluded that the results were not sugges-tive of the high relasugges-tive risks of cancer reported for TCDD in previous studies The slight risk of increased soft tissue sarcoma is weakened by the small numbers involved (only three cases) and confounding factors such as smoking and exposure to other chemicals

Figure 22 Effect of TCCD on the (simplified) heme synthetic pathway.

GLYCINE + SUCCINYL CoA

GAMMA AMINOLEVULANIC ACID ( ALA )

PROTOPORPHYRIN IX

HEME ( feedback inhibitor )

HEME SYNTHASE VARIOUS STEPS ( uroporphyrinogens, coproporphyrinogens )

ALA Synthase ( TCDD increases levels )

Fe ++

In another epidemiologic study of 754 Monsanto employees exposed to high levels of TCDD in a 1949 accident, of whom 122 developed chloracne, there was no increased incidence of cancer in those who developed chloracne, although they were presumably the group with the highest exposure Con-versely, workers who were also potentially exposed to 4-aminobiphenyl, a potent bladder carcinogen, had increased mortality from bladder cancer, lung cancer, and soft tissue sarcoma This suggests that TCDD might act as a co-carcinogen or promoter Again, the effects of confounders such as smoking and exposure to other chemicals could not be ruled out, but recent experi-mental evidence supports the suspicion that TCDD could act in this way Walsh et al studied the cell toxicity of aflatoxins in cultured human epidermal cells AFB1 was markedly toxic at 1 µg/mL Neither AFB2 nor AFB1 dihydrodiol were toxic TCDD alone was not toxic to the cells but at

5 nM, it dramatically stimulated AFB1 toxicity at levels as low as 0.1 µg/mL

It also increased the formation of AFB1 epoxides and a 20-fold increase in DNA adduct formation was observed AFB1 is the most carcinogenic of the aflatoxins (see Chapter 10)

The most recent report from Seveso is suggestive of a carcinogenic effect

in humans The exposed population was divided into three groups according

to their likely level of exposure Persons who had left the area were traced with a 99% success rate They were followed from 1977 to 1986 and cancer incidences were compared to a reference population not exposed to high TCDD levels The population exposed to the highest levels was small, but nearly 5000 were in the middle exposure area In these, the relative risk factors compared to the reference population were elevated for several cancers: 2.8 for hepatobiliary cancer, 5.7 for lymphoreticulosarcoma in men, 5.3 for multiple myeloma, and 3.7 for myeloid leukemia in women In the lowest exposure zone, the incidence of non-Hodgkin’s lymphomas and soft tissue tumors was elevated, especially among those who had lived in the area for over 5 years Paradoxically, the incidences of breast cancer and endometrial cancer were below expected levels

Weaknesses in this study include the inability to control for confounding factors such as smoking and the lack of hard data regarding real exposure levels Despite the conflicting results of several epidemiological studies, most authorities now agree that there is a high index of suspicion for TCDD carcinogenicity in humans, especially for non-Hodgkin’s lymphoma The definitive word, however, remains to be heard, and the existing evidence comes from high industrial and occupational exposures that may not be relevant to environmental exposures encountered by the general population

if a threshold truly exists because of the TCDD receptor story (see next section and Figure 23)

The mechanism of carcinogenesis in animals appears to be epigenetic Recent evidence indicates that TCDD binds to a specific receptor, the Ah (for aryl or aromatic hydrocarbon) receptor, and that a minimum number

of Ah receptors must be occupied for TCDD to exert its effect (see below) The implication of this is that there is a threshold dose and that the linear

multistage carcinogenesis model is inappropriate for TCDD and for any other agent, such as PCDDs and PCDFs, that works by this mechanism (see Chapter 3 and below) The EPA is now reconsidering the use of the linear multistage model, at least for TCDD and perhaps for a few other agents

Neurotoxicity

Many toxic effects have been observed, including impaired vision, hearing, and smell, depression, sleep disturbances, and others These were observed

in workers exposed to TCDD in the 1949 Monsanto accident

Reproductive toxicity

Testicular atrophy, necrosis, and decreased spermatogenesis have been seen

in laboratory animals

Metabolic disturbances

Weight loss and depletion of adipose tissue occur in lab animals

The role of the aryl hydrocarbon receptor (AhR) and enzyme induction

TCDD is one of the most potent inducers of aryl hydrocarbon hydroxylase (AHH) yet discovered In some species it is effective at 1 µg/kg It induces synthesis of hepatic microsomal cytochrome P450 (CYP1A1 and possibly 1B1) TCDD uptake into the cell is passive (i.e., concentration gradient depen-dent) Intracellularly, it binds to the aromatic hydrocarbon cytosolic receptor (AhR), which is the product of a regulatory gene The unliganded aryl hydrocarbon receptor complex (AhRC) contains the aryl hydrocarbon recep-tor (AhR) Binding of an aromatic hydrocarbon ligand causes release of the AhR that is translocated to the nucleus by a translocator protein The unli-ganded AhRC is heteomeric; but after binding to a halogenated aromatic hydrocarbon (HAH) or a polycyclic aromatic hydrocarbon (PAH), the AhR

is released and associates with the AhR nuclear translocator protein to form heterdimeric transformed AhRC Ligands for the AhR are typically hydro-phobic aromatic compounds, including the HAHs dibenzo-p-dioxins, diben-zofurans, biphenyls, and PAHs such as benzo[a]pyrene and many other benzo derivatives (products of combustion), naphthalene, naphthacene, and many others (see http://chrom.tutms.tut.ac.jp/JINNO/DATA-BASE/00alphabet.html)

In the nucleus, AhRC activates numerous xenobiotic-responsive struc-tural genes The information is transcribed to m-RNA and translated to protein synthesis and the production of cytochrome P450 (1A1, 1A2, 1B1) This is known as a pleiotropic response; that is, it results in more than one phenotypic effect TCDD is the most potent known inducer of AHH The consequence of this induction is that several drug (xenobiotic-)metabolizing enzymes are induced, and reactive metabolites may be formed that react with proteins and nucleic acids to cause mutations, teratogenesis, and

carcinogenesis, as well as altered drug metabolism Conversely, the reactive metabolites may be excreted or detoxified, as by conjugation with glucu-ronide A schematic representation of this pathway is shown in Figure 23

Figure 23 The Ah receptor and enzyme induction.

NUCLEUS

CELL MEMBRANE PASSIVE DIFFUSION BINDING TO Ah RECEPTOR

INDUCER-RECEPTOR COMPLEX TRANSLOCATED TO NUCLEUS

ACTIVATION OF VARIOUS STRUCTURAL GENES

TRANSCRIPTION

TO mRNA TRANSLATION TO PROTEIN SYNTHESIS {eg ARYL HYDROCARBON HYDROXY-LASE, MEMBRANE-BOUND

CYP-450}

INCREASED BIOTRANSFORMATION OF

XENOBIOTICS REACTIVE METABOLITES MAY CAUSE

MUTAGENESIS, CARCINOGENESIS,

TERATOGENESIS, ALTERED DRUG

METABOLISM

EXTRACELLULAR AROMATIC INDUCER eg TCDD

This system has been most studied in the mouse, where it is inherited as an autosomal dominant pattern Similar systems have been identified in the rat, the rabbit, and some fish It is most heavily concentrated in the liver In humans, there is considerable variation in the Ah locus To date, no endog-enous substrate has been identified for the Ah receptor AhR “knockout” mice, however, have small, fibrosed livers In the absence of an exogenous ligand, AhR has been found in the nucleus of cultured Hela cells, behaving

as if they were ligand bound It seems apparent that the AhR has an impor-tant physiological role It is known that primitive species (bacteria, yeasts) utilize polycyclic hydrocarbons as an energy source and possess P450 metab-olizing enzymes for them (camphor in Pseudomonas, benzo[a]pyrene in yeast), so these may have evolved as a detoxication system The mechanism

of TCDD toxicity is not known, but if there is a natural substrate for Ah receptors, its displacement by TCDD could be involved in the latter’s toxicity

It is not clear whether TCDD itself or its metabolite(s) are responsible TCDD also has non-receptor-mediated effects, including interference with calcium homeostasis and a variety of membrane-related changes It is interesting that the chloracne associated with TCDD also occurs with bro-mides, which could not act as Ah ligands For more on TCDD toxicity, see Chapter 12

Paraquat toxicity

This PAH herbicide (1,1′-dimethyl-4,4′-bipyridinium ion) is highly water soluble and therefore poorly absorbed across the skin or gastrointestinal mucosa It is extremely toxic to humans when inhaled, however, and 5 g may be fatal Pulmonary congestion, edema, and hemorrhage may result in almost complete functional destruction of the lung In severe cases, lung transplantation has been tried as a last resort, with disappointing results Although paraquat is poorly absorbed from the oral route, its highly toxic nature can result in lung toxicity days or weeks later It is thus one of the

“hit-and-run” class of toxicants Liver and kidney damage and neurological damage also occur (see Chapter 9 on pesticides)

Insecticides

Chemical insecticides are used to increase food crop production, protect livestock and household pets against insect pests (warble fly, bot fly, screw-worm, fleas), control disease-carrying insects (anopheles mosquitoes that carry malaria), and control destructive insects such as termites It was the chlorinated hydrocarbon insecticide DDT (dichlorodiphenyltrichloroethane) that first raised concerns over the impact of pesticides on the environment

In 1961, the author Rachael Carson brought out her book Silent Spring, in which she documented the devastating effect that this chemical had on bird life because it weakened eggshells so that the eggs collapsed in the nest or the chicks were abnormal at hatching The persistence of the chemical (it

and its metabolite DDE have a biological t1/2 of 50 years) and its high lipid solubility result in its biomagnification up the food chain

Predatory and fish-eating birds are especially vulnerable to DDT The product was banned in the United States and Canada in 1972, but it is still used elsewhere in the world, including Mexico, and trace levels may be present in imported products Levels of from 1 ppb to 1 ppm may be present

in fish, oysters, and other seafoods and can contribute to human tissue levels

of up to 10 ppm

Halogenated hydrocarbon insecticides (chlorinated hydrocarbons) are principally neurotoxic, interfering with axonal transmission by altering sodium and potassium transport across the axonal membrane to prevent normal repolarization Evidence of carcinogenicity also has been obtained

in animal experiments (see Chapter 9 on pesticides for more details)

Industrial and commercial chemicals

Biphenyls

Polybrominated biphenyls (PBBs) are used as fire retardants in thermoplas-tics for TV and office machine casings The more familiar polychlorinated biphenyls (PCBs) are highly stable and resistant to degradation in acids or bases, by oxidation or heat (to 800°C)

These characteristics make PCBs ideal insulators in electrical transform-ers, as hydraulic fluid, and in brake linings They are also used as plasticizers

in polymer films The same characteristics, however, make these agents very persistent in the environment Exposure to these compounds is largely an occupational hazard, but exposure in the environment can occur as a result

of contamination of groundwater from spills, improper storage of waste PCBs from old transformers and capacitors, or fires in storage sites Although the manufacture of PCBs was banned in the United States in 1977, they remain a problem because of their persistence and resistance to destruction Forty percent of North Americans have body fat levels of 1 ppm or higher (these agents have high lipid solubility) Prior to 1970, 500,000 tons of PCBs were produced in North America

Toxicity

1 Animal The LD50 in rats may be 1 to 10 g/kg Chronic toxicity in-volves skin lesions, hepatotoxicity, immunosuppression, and repro-ductive dysfunctions Carcinogenicity has also been reported Re-cently, genetic damage in cetaceans (whales, dolphins) and seals in the Baltic Sea has been ascribed to high levels of PCBs

2 Human Characteristic chloracne, impaired immune response, liver damage, gastrointestinal disturbances (nausea, vomiting, loss of ap-petite), and CNS disturbances (weakness, ataxia) as well as repro-ductive problems and cancer have been associated with exposures